- Volume 73, Issue 12, 1992
Volume 73, Issue 12, 1992
- Review Articles
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Bluetongue virus proteins
More LessIntroduction. Bluetongue virus (BTV) is the prototype virus of the Orbivirus genus in the Reoviridae family. Orbiviruses that infect and are transmitted by arthropod vectors (e.g. gnats, ticks, mosquitoes, etc.) include viruses that may cause disease in their vertebrate hosts with serious economic consequences in some regions of the world. The BTV group, which consists of at least 24 different serotypes (BTV-1, -2, etc.), infects field and domestic animals (e.g. sheep and cattle), occasionally with high morbidity and mortality but often with almost no apparent clinical symptoms. Other orbiviruses that may cause disease in animals include African horse sickness virus (AHSV; nine serotypes, AHSV-1, -2, etc.) and epizootic haemorrhagic disease virus (EHDV; seven serotypes, EHDV-1, -2, etc.) of deer. Although BTV and AHSV were isolated in 1900, and initial morphological and biochemical characterization were reported as early as 1969 (see review article, Gorman, 1990), much of the current knowledge on the molecular biology, genome structure and encoded products has been obtained only recently. The data are predominantly based on research on BTV.
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The molecular biology of poliovaccines
More LessThe molecular biology of the live attenuated Sabin vaccine strains of poliovirus has been studied extensively, and surprisingly few mutations are required to account for the greater part of the attenuated phenotype. The viruses are clearly capable of extremely rapid, extensive and precise variation in the vaccinee to adapt from the attenuated form to a form able to grow successfully in the host, yet despite this they cause almost no disease. The high degree of genetic variation in the face of general phenotypic stability in the wild-type suggests that polioviruses are extremely well adapted to their hosts and that vaccines exploit some aspects of the virus host ecology to be safe and effective. The precise mechanisms by which they do so raise possibilities of improving vaccine production and testing methods, and designing better vaccine strains.
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Distinct signals in human immunodeficiency virus type 1 Pr55 necessary for RNA binding and particle formation
More LessThe human immunodeficiency virus type 1 (HIV-1) gag gene product Pr55 self-assembles to form virus-like particles when expressed in Spodoptera frugiperda cells using recombinant baculoviruses. The particles resemble immature HIV and are released from the infected cell into the culture medium. Using this system we have progressively truncated the gag open reading frame from the C terminus and examined each deleted gag protein for its particle-producing capability. We show that deletion of Pr6 and deletions that progressively remove the distal region of the Pr7 domain, including one Cys-His box thought to function as an RNA capture signal, do not affect particle formation. However deletion of two Cys-His boxes causes production of slightly larger particles with altered sedimentation properties. Sequence-specific North-Western assays using an RNA probe representative of the HIV-1 packaging signal revealed specific RNA binding by all mutants that maintained both Cys-His boxes. However, deletion of one Cys-His box reduced RNA binding substantially and loss of two Cys-His boxes abolished binding entirely. We conclude that HIV-1 gag particle formation per se does not require viral RNA encapsidation, but that it may act as a cofactor in the condensation of the immature core. Further deletion of gag sequences upstream of the Cys-His boxes led to the abolition of particle-forming ability, and we show that one boundary of the gag sequence necessary for particle formation lies within eight amino acids spanning one of the known protease cleavage sites at the C terminus of Pr24.
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Extrachromosomal human immunodeficiency virus type 1 DNA forms in fresh peripheral blood lymphocytes and in two interleukin-2-independent T cell lines derived from peripheral blood lymphocytes of an asymptomatic seropositive subject
Two immature T cell lines (FT1 and FT4) were established after in vitro cloning of peripheral blood lymphocytes (PBLs) from an asymptomatic human immunodeficiency virus type 1 (HIV-1) seropositive, human T cell-lymphotropic virus type 1 seronegative homosexual subject. Although derived from a limiting dilution cell cloning assay, these cell lines were not recloned for this study. Their growth was independent of exogenous interleukin-2. Both cell lines were able to form colonies when cloned in agar, but failed to form solid tumours when injected into nude mice. FT lines belong to the very immature T cell lineage as they exhibit rearranged TCR genes but no expression of T cell membrane antigens, including CD2, CD3, CD4, CD6, CD7 and CD8. They also contain an HIV-1 genome that was detected only in an extra-chromosomal DNA form, even after several passages in vitro. The presence of unintegrated viral DNA was also detected by polymerase chain reaction analysis in the same sample of fresh uncultured PBLs. Furthermore, despite the absence of CD4 expression, both T cell lines were susceptible to CD4-independent HIV-1 superinfection (lack of superinfection inhibition in the presence of OKT4A monoclonal antibodies).
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Carbohydrate determinant NeuAc-Galβ(1-4) of N-linked glycans modulates the antigenic activity of human immunodeficiency virus type 1 glycoprotein gp120
In the present study we investigated to what extent the peripheral carbohydrate structure of N-linked glycans influences the antigenic properties of human immunodeficiency virus type 1 glycoprotein 120 (gp120). Recombinant gp120 was purified from GMK cells infected with a recombinant vaccinia virus expressing gp120. Purified gp120 was then coated onto 96-well ELISA microplates and subjected to sequential removal of peripheral monosaccharide units. Modified or unmodified gp120 was then incubated with monoclonal antibodies recognizing specific epitopes of gp120 and with a reporter lectin to determine the extent of carbohydrate elimination. Antibody and lectin binding was quantified in an enzyme-linked system. We found that the carbohydrate structure NeuAc-Galβ(1-4) of N-linked glycans, defined both by lectin reactivity and by specific glycosidases, is involved in modulating the binding of antibody to a number of epitopes of peptide nature. The binding of antibody to one class of epitopes, situated in a region between amino acids 200 and 230, was strongly increased by removal of NeuAc-Galβ(1-4). whereas the binding to epitopes in the V3 region was decreased and the binding to epitopes in the far N-terminal region was not altered by the treatment. These results suggested that peripheral structures of N-glycans are involved in modulating the overall conformation of gp120.
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Strain-specific selection of genome segments in avian reovirus coinfections
More LessTo determine whether selection of genome segments in coinfections is strain-specific, chicken embryo fibroblasts were coinfected with avian reovirus strain 883 and one of three other avian reovirus strains (176, S1133 and 81-5). Viral progeny from each coinfection (883 × 176, 883 × S1133 or 883 × 81-5) was serially passaged at a low m.o.i. The electropherotypes of the coinfection progeny and those of the plaque-derived clones obtained from passages 1 and 20 were analysed. Two 883 segments (M2 and S2) were found to be selected in the 883 × 176 coinfection, three 883 segments (M2, M3 and S2) in the 883 × S1133 coinfection, and only one 883 segment (M3) in the 883 × 81-5 coinfection, i.e. different 883 genome segments were selected in the three coinfections. It was, therefore, concluded that selection of genome segments in a coinfection of a given cell line is virus strain-specific. The selection of genome segments in coinfections was shown to be due to enhanced infectivity of the reassortants that were formed in the coinfections. In addition, defective interfering particles that lack the S1 segment were identified in the 883 × 81-5 coinfection progeny following serial passage. Selection of genome segment(s) in coinfections as described herein may have potential importance on the effect and production of divalent or multivalent vaccines.
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Nucleotides 9 to 11 of the influenza A virion RNA promoter are crucial for activity in vitro
More LessThe 12 nucleotide conserved sequence at the 3′ end of influenza A virion RNA is sufficient to function as a promoter in vitro. By introducing point mutations in all 12 positions of this promoter in model RNA templates and studying the efficiency of RNA synthesis in vitro, we show that only three nucleotides, residues 9, 10 and 11, are crucial for activity, although other nucleotides play a significant but less important role. Additions or deletions within the promoter are tolerated, resulting in either an increase or a decrease in promoter activity, depending on the mutation introduced; in some cases premature termination is caused. Taking these observations into account, a model for RNA polymerase binding and copying of the promoter is discussed.
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Restricted replication of vesicular stomatitis virus in T lymphocytes is coincident with a deficiency in a cellular protein kinase required for viral transcription
More LessVesicular stomatitis virus (VSV) fails to replicate in mouse T lymphocytes unless the cells have been mitogenically stimulated with concanavalin A (Con A). We have examined the possibility that the failure of VSV to replicate in unstimulated T lymphocytes can be attributed to a deficiency in a host protein kinase which activates the viral P protein by phosphorylation, thus rendering it transcriptionally competent. Soluble extracts were prepared from purified mouse T lymphocytes, with or without prior treatment with Con A. The ability of these extracts to phosphorylate bacterially synthesized P protein of two VSV serotypes was measured in vitro. Activity of the protein kinase on the P proteins of the Indiana or New Jersey serotypes of VSV increased, on average 2.4- and 2.1-fold respectively, after treatment of the cells with 3 µg/ml Con A. Higher concentrations of Con A induced proportional increases (up to 10-fold) in the activity of the host protein kinase. Activities of the kinase phosphorylating the P protein in separate populations of CD4- and CD8-containing murine T lymphocytes increased similarly on mitogenic activation. No biochemical or immunological differences were observed between the T cell protein kinase and the previously characterized protein kinase (casein kinase II) from BHK-21 cells. The activity of the kinase that phosphorylates the P protein did not vary in CV-1 cells on treatment with α- or γ-interferon, both of which inhibited VSV replication. Similarly, casein kinase II activities in Raji and SIRC cells, which do not normally support VSV growth, were the same as in BHK-21 cells. Thus restriction of VSV replication in these cells, in contrast to T lymphocytes, was not associated with a deficiency in the host casein kinase II activity.
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Activation of protein kinase C inhibits adenovirus VA gene transcription in vitro
More LessWe report here that activation of protein kinase C (PKC) results in the inhibition of adenovirus virus-associated (VA) gene transcription in vitro. The involvement of PKC in this inhibition is supported by the fact that the addition of PKC inhibitors to transcription reactions in which the PKC cofactor phosphatidyl serine (PS) was present resulted in increased levels of transcription compared to those in reactions in which PKC activity was stimulated in the absence of PKC inhibitors. Furthermore, based on these in vitro studies we propose that the inhibition of VA gene transcription is possibly due to the inability of the VA gene to form an active transcription complex following the activation of PKC. This conclusion was drawn from in vitro data which demonstrated that PS stimulation of endogenous PKC present in cell extracts resulted in failure to isolate a fully initiated, stable transcription complex.
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Molecular basis of hepatitis B virus serotype variations within the four major subtypes
More LessAmino acid residues 101 to 180 of hepatitis B surface antigen (HBsAg) were predicted by sequencing the corresponding part of the S gene of hepatitis B virus (HBV) DNA in 46 HBsAg-positive sera, which had been subtyped by immunodiffusion with respect to d/y, w/r, w1 to w4 and q. The sequences of the nine different HBV serotypes defined by these specificities were found to be homogeneous proving that they represent consistent variations of HBV at the genomic level. Residue 127 was found to be important as were Pro, Thr and Leu for w1/w2, w3 and w4, respectively. Five residues were found to differ between ayw1 and ayw2. These were at positions 134 (Phe instead of Tyr), 143 (Thr instead of Ser), 159 (Ala instead of Gly), 161 (Tyr instead of Phe) and 168 (Val instead of Ala). However, all these residues were shared by ayw1 and adw2, implying that Arg122 was also important for w1 expression. All genomes expressing r, apart from one ayr strain, had an Ile126, which might explain the pseudo-allelism of w1 to w4 in relation to r, since this substitution might influence the w epitope. There were two regions where adw4q − and adrq − differed from all the q + subtypes. These were located at residues 158 and 159, and at residues 177 and 178, where both the q − subtypes had amino acid substitutions in adjacent positions. The mapping of the epitopes defining these antigenic specificities will help to link information on the world-wide distribution of HBsAg subtypes to future molecular epidemiology with regard to HBV.
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Rearrangements of the upstream regulatory region of human papillomavirus type 6 can be found in both Buschke-Löwenstein tumours and in condylomata acuminata
Clinically malignant Buschke-Löwenstein tumours and benign condylomata acuminata are caused by human papillomaviruses (HPVs), predominantly HPV-6 and -11. In some cases, the HPV-6 genomes found in Buschke-Löwenstein tumours and in verrucous carcinomas differ from HPV-6b isolated from a benign genital wart, by rearrangements of the upstream regulatory region (URR). To evaluate the frequency and role of mutations of the URR of HPV-6 we analysed 42 condylomata acuminata and four Buschke-Löwenstein tumours by the polymerase chain reaction and restriction enzyme cleavage. Using only four different restriction enzymes we could demonstrate four distinct restriction patterns, indicating that naturally occurring HPV-6 isolates display a high degree of DNA polymorphism within the URR. One Buschke-Löwenstein tumour and two condylomata acuminata yielded rearranged URRs with DNA duplications. All three lesions harboured multiple HPV-6 variants, suggesting that cellular or environmental factors facilitate the development of rearrangements. Therefore, rearrangements of the URR may represent only secondary events in condylomata acuminata and Buschke-Löwenstein tumours which do not necessarily confer a higher malignant potential to the infected cell.
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Phenotypic characterization of three temperature-sensitive mutations in the vaccinia virus early gene transcription initiation factor
More LessVaccinia virus gene D6R encodes the small subunit of the virion early gene transcription initiation factor. Three temperature-sensitive mutations have been mapped to this gene. The biochemical phenotype exhibited by each mutation was examined. All mutants displayed altered viral protein synthesis in pulse-labelling analyses at both the permissive and non-permissive temperatures. The onset of early protein synthesis was delayed, and the rate of early protein synthesis was reduced in each case. Furthermore the shut-off of both host and early protein synthesis was delayed. In pulse-chase experiments, the stability of the D6R protein in E93- or C46-infected cells was shown to be reduced at 40°C relative to that at 31°C. Early mRNA was quantified in cells at 2 h post-infection and shown to be reduced substantially. The ability of each mutant virus to support transcription in vitro was examined at both temperatures and, of the three mutants, only S4 transcription was shown to exhibit reversible temperature sensitivity.
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Expression of genes for the Epstein—Barr virus small RNAs EBER-1 and EBER-2 in Daudi Burkitt's lymphoma cells: effects of interferon treatment
More LessThe relative levels, rates of synthesis and stabilities of the abundant Epstein—Barr virus (EBV)-encoded small RNAs, EBER-1 and EBER-2, were examined in Daudi Burkitt's lymphoma cells. Although both RNAs are transcribed at approximately equal rates, the steady-state level of EBER-1 is at least 10-fold greater than that of EBER-2. This is shown to be due to a much faster rate of turnover of EBER-2. In the presence of actinomycin D, the half-lives of EBER-1 and EBER-2 are 8 to 9 h and 0.75 h, respectively. Following treatment of the cells with human interferon (IFN) α the transcription of both RNAs is strongly inhibited. However, the level of EBER-1 increases up to twofold, indicating a further stabilization of this RNA. In IFN-treated cells, EBER-2 accumulates in the form of truncated products. Nuclease protection experiments indicated that this is due to a post-transcriptional modification of the 3′ end of the molecule. These data show that the effects of IFN treatment on the expression of these two viral gene products are very complex in cells latently infected with EBV.
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Characterization of the nucleotide sequence of the Lymantria dispar nuclear polyhedrosis virus DNA polymerase gene region
More LessThe DNA polymerase gene of the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus (LdMNPV) was cloned and sequenced. The predicted DNA polymerase protein (1113 amino acids, 115.9K) was found to have an amino acid identity of 48% with the corresponding gene of the Autographa californica MNPV (AcMNPV). It contains five domains associated with substrate binding, primase interaction, and pyrophosphate hydrolysis and three domains associated with 3′»5′ exonuclease activity common to other DNA polymerases. A region with a conserved TATA promoter and a CAGT mRNA start site sequence motif was identified and shown to be transcribed by RNA polymerase II, indicating that the LdMNPV DNA polymerase gene is expressed as an early gene. An open reading frame possibly expressed as a late gene, oriented in the opposite direction and overlapping the N-terminal coding region of the DNA polymerase gene was found in the LdMNPV sequence and was shown to be conserved in the same position in AcMNPV.
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Equilibrium and kinetic analysis of Autographa californica nuclear polyhedrosis virus attachment to different insect cell lines
More LessThe kinetic and equilibrium attachment of Autographa californica nuclear polyhedrosis virus (AcMNPV) to seven insect cell lines was evaluated. Kinetic experiments revealed differences of up to 10-fold in the infection rates among cell lines. Equilibrium binding also varied between cell lines and was saturable. The Tn 5B1-4 and Tn F cell lines had the highest virus binding affinities and infection rates and exhibited diffusion-limited attachment. The rate of infection appears to be limited by the rate of attachment. For the Tn 5B1-4 cells the physical to infective particle ratio for AcMNPV was 5.3. From the Scatchard analyses, the cell lines Tn 5B1-4 and Tn F displayed affinities of 2.35 × 1010 m −1 and 1.60 × 1010 m −1, respectively, with 6000 and 13700 binding sites per cell. The insect cell line Hz 1075, which is not susceptible to AcMNPV infection, displayed a much lower, but saturable, binding of AcMNPV with 900 sites/cell and an affinity of 1.1 × 1010 m −1. Unlabelled AcMNPV, but not Lymantria dispar MNPV could compete with labelled AcMNPV for binding sites. There were 93 to 96% reductions in virus cell binding following pretreatments of cells with three proteases, suggesting the involvement of a cellular protein component in virus binding. Tunicamycin, an inhibitor of N-linked glycosylation and expression of some membrane proteins on the cell surface, reduced virus binding in a dose-dependent manner suggesting a role for glycoprotein(s) in binding. However there was no evidence for the direct involvement of oligosaccharides in attachment. Metabolic inhibitors of oligosaccharide trimming and competition binding assays using simple sugars caused no measurable reductions in virus binding. These findings suggest that AcMNPV attachment to insect cells is receptor-mediated via a glycoprotein component(s); the direct involvement of oligosaccharide moieties in binding is unlikely.
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Protein synthesis in pupae of the silkworm Bombyx mori after infection with nuclear polyhedrosis virus: resistance to viral infection acquired during pupal period
More LessProtein synthesis has been studied in pupae of the silkworm Bombyx mori (Bm) infected with nuclear polyhedrosis virus (BmNPV) at various stages of the pupal period. Nascent proteins were labelled by injection of [35S]methionine into pupae and then analysed by SDS-PAGE. Temporal regulation of synthesis of infected cell-specific proteins (ICSPs) in pupae was demonstrated by electrophoretic analysis of the proteins labelled at different times post-infection (p.i.). The rate of ICSP synthesis reached a maximum at 4 to 5 days p.i., exceeding the rate of synthesis of cellular proteins in uninfected pupae by about twofold. The viral proteins p10 and polyhedrin were the most abundant products synthesized late in the infection. Both proteins were found to be associated with the nuclear matrix after fractionation of nuclei from infected pupae. Two virus-induced phosphoproteins, pp35 and ppB, were found to be the major acceptors of labelled phosphate from [γ-32P]ATP during in vitro phosphorylation of proteins in pupal homogenates, nuclei and nuclear extracts. These proteins had electrophoretic mobilities comparable to those of structural phosphoproteins of BmNPV virions with M rs of 35K and 11K to 16K, respectively. The latter polypeptide was identified as the major DNA-binding protein of the virus. The susceptibility of silkworms to BmNPV decreased markedly during the pupal period. Following injection of BmNPV all young pupae acquired polyhedrosis and finally died whereas most of the older pupae did not exhibit disease and completed metamorphosis normally. Moreover, the later in the pupal period the silkworms were infected, the lower the production of polyhedrin in diseased pupae.
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Induction of a novel protein kinase in pupae of the silkworm Bombyx mori after infection with nuclear polyhedrosis virus
More LessProtein kinases induced by Bombyx mori nuclear polyhedrosis virus in pupae of the silkworm B. mori were examined by activity gel analysis using phosvitin as a protein substrate. The method involved PAGE of the soluble fraction from pupae under native conditions and in the presence of SDS, followed by in situ renaturation of proteins and recovery of protein kinase activity in the intact gel. A novel protein kinase able to phosphorylate phosvitin was detected in the infected pupae from 2 days post-infection. This enzyme was not present in uninfected silkworms at any stage of the pupal period. The novel kinase activity was found by SDS-PAGE to be associated with a single polypeptide with an apparent M r of 50K. However, on electrophoresis under native conditions its activity was associated with a set of polypeptides with similar but not identical electrophoretic mobilities. Microheterogeneity of the catalytically active polypeptides suggests that the virus-induced protein kinase undergoes post-translational modification during the course of infection.
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Comparison of the thymidine kinase genes from three entomopoxviruses
More LessThe entomopoxviruses (insect poxviruses) of eastern spruce budworm (Choristoneura fumiferana), two year cycle spruce budworm (C. biennis) and the Indian red army worm (Amsacta moorei) are being studied in our laboratory for their potential as biological insecticides and expression vectors. These viruses characteristically replicate in the cytoplasm of insect cells and produce occlusion bodies that serve to protect the virion from the environment. By analogy to mammalian pox-viruses, they should also contain a viral thymidine kinase (TK) that functions in viral DNA synthesis. The replication of the A. moorei entomopoxvirus was inhibited by bromodeoxyuridine whereas the baculovirus of Autographa californica was insensitive to this drug. This result was a biochemical indication that entomopoxviruses contained a kinase that phosphorylated this nucleoside analogue and thus viral DNA synthesis was inhibited. TK genes from the three different insect poxviruses were identified, cloned and sequenced. The sequences of the TK genes of the entomopoxviruses were closely related and exhibited 63.2% identity and 9.9% similarity at the protein level. However, there was only 36.7% identity and 13.6% similarity when these enzymes were compared to their mammalian poxvirus counterpart in vaccinia virus. Finally, one entomopoxvirus TK gene was expressed in Escherichia coli mutants lacking the enzyme. These bacteria were converted to a phenotype that could incorporate radioactive thymidine into their chromosomal DNA. The results presented in this paper provide impetus for the design of a recombinant entomopoxvirus expression system in which foreign genes could be introduced into the viral TK locus under selective pressure from bromodeoxyuridine.
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A myxoma virus intergenic transient dominant selection vector
More LessThe purpose of this study was to construct an intergenic transfer vector which can be used for the generation of recombinant myxoma viruses (MVs) expressing a foreign gene insert. Recombinant MVs expressing the Escherichia coli lacZ gene were constructed in vitro by transfection of MV-infected rabbit cells with a transfer expression vector, and isolated under growth conditions selecting for transient expression of the E. coli gpt gene. The effect of inserting foreign DNA sequences between the viral thymidine kinase gene and open reading frame MF8a upon the transcription of these genes was investigated.
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On the structure, genesis and significance of DNA duplications at the Rous sarcoma proviral insertion sites in Rat-1 cells
More LessWe have previously shown that most Rous sarcoma proviruses in Rat-1 cells are simple insertions, at apparently random sites, and are not transcribed. A small minority of simple insertions are transcribed and these show a bias to insertion at sites closely 3′ to presumptive cellular CG-rich islands. However, most transcribed proviruses are complex, typified by contiguous duplications of proviral DNA 5′ to a complete proviral unit. The cellular sites of these complex insertions are unknown and their structure and significance incompletely documented. We report here more extensive analyses of proviral duplications, with the following findings. The 5′ duplications predominantly involve proviral regions known to contain enhancer functions, substantiating earlier data. The cellular insertion sites are not biased to CG-rich loci at the level displayed by simple transcribed proviruses. The detailed structure of two duplications, and partial analysis of several others, strongly favours their genesis by illegitimate template transfer at reverse transcription, followed by self-recombination. These findings show that aberrant reverse transcription can generate duplications that dispense with the need for an expressed provirus to be integrated at a favourable cellular site.
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The efficiency of cell targeting by recombinant retroviruses depends on the nature of the receptor and the composition of the artificial cell-virus linker
More LessUsing streptavidin-bound antibodies specific for both viral and cell membrane epitopes, we have reported previously that human cells may be infected by murine ecotropic retroviruses through an interaction with major histocompatibility complex class I and class II antigens, and thus have demonstrated that cell targeting by recombinant retroviruses is feasible. We report here that (i) growth factor or hormone receptors, such as those for epidermal growth factor (EGF) and insulin, can also mediate infection of human cells; (ii) a biotinylated cytokine or hormone can substitute for the anti-cell antibody in bispecific antibody complexes, thus extending the versatility of the method; (iii) although yields are low in our assay, infection efficiency clearly appears to depend upon the biochemical composition of molecular bridges because bi-functional antibody complexes are more efficient than cytokine-antibody complexes in the case of the EGF receptor. Finally, our study indicates that different cell membrane molecules are not equally efficient in allowing infection of human cells because targeting of the transferrin, high density lipoprotein and galactose receptors, as well as that of various membrane glycoconjugates, by murine ecotropic retroviruses did not lead to the establishment of a proviral state.
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Human immunodeficiency virus type 1-infected HL-60 cells are capable of both monocytic and granulocytic differentiation
More LessWe have used the human myelomonocytic cell line HL-60 as a model system to determine whether human immunodeficiency virus type 1 (HIV-1) infection affects differentiation of myeloid progenitor cells. HL-60 cells were infected with three HIV-1 isolates (IIIB, NL4-3 and PM213). HIV-1 antigen expression and cytopathicity in HL-60 cells infected with each of the three isolates was delayed by approximately 15 days as compared to those in the prototypic T cell line, H9. Chronically infected HL-60 cells and clonal lines derived from them were treated with dimethyl formamide (DMF) and induced to differentiate into granulocytes. Approximately the same percentage of these cells as of DMF-treated, uninfected HL-60 cells differentiated. Superoxide production by infected and uninfected DMF-induced cells was similar. Likewise, approximately the same percentage of cells in infected and uninfected cultures became adherent and were positive for non-specific esterase when monocytic differentiation was induced. The data demonstrate that HL-60 cells infected with HIV-1 are capable of morphological and functional granulocytic and monocytic differentiation.
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Detection and typing of human papillomaviruses present in fixed and stained archival cervical smears by a consensus polymerase chain reaction and direct sequence analysis allow the identification of a broad spectrum of human papillomavirus types
DNA well suited for polymerase chain reaction (PCR) amplification was purified from archival Papanicolaou smears. The detection of a wide range of human papillomavirus (HPV) types was made possible using a HPV-specific consensus primer pair, and typing was conveniently done by direct sequence analysis of the PCR product. The method could be of unique value in longitudinal and cross-sectional studies aimed at answering a number of fundamental pathological and epidemiological questions regarding HPV infection of the genital tract.
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In vitro infection of normal human keratinocytes by human papillomavirus type 1 followed by amplification of the viral genome in reconstructed epidermis
Primary cultures of normal human keratinocytes were inoculated in vitro with human papillomavirus type 1 (HPV-1), the agent responsible for deep plantar warts. Upon transfer to dead de-epidermized dermis and growth at the air-liquid interface, keratinocytes reconstituted a pseudoepidermis. Under these highly differentiating conditions, HPV-1 DNA amplification was found to take place in the reconstructed epidermis, being detectable from 7 days after the transfer and persisting for at least 10 days thereafter. The extent of keratinocyte differentiation may be insufficient to allow a complete HPV infectious cycle.
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Murine gammaherpesvirus 68 establishes a latent infection in mouse B lymphocytes in vivo
More LessMurine gammaherpesvirus 68 (MHV-68) is able to persist in spleen cells of infected mice. To determine the cell type harbouring persistent virus, spleen cells from infected animals were separated into immunoglobulin (Ig)-positive (B cell-enriched), Ig-negative (T cell-enriched) and plastic-adherent (macrophage-enriched) fractions. These cells were co-cultivated with permissive BHK-21 cells in an infectious centre assay. The consistent recovery and enrichment of infectious centres in the Ig-positive fraction clearly demonstrates that B cells are a major site of virus persistence/latency. This observation indicates that MHV-68 is biologically similar to Epstein—Barr virus and other members of the B cell lymphotropic gammaherpesvirus 1 subgroup.
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Primary chimpanzee skin fibroblast cells are fully permissive for human cytomegalovirus replication
More LessCytomegaloviruses generally display a host range restricted to differentiated cell types from the species they infect. For human cytomegalovirus (HCMV) this has meant that with few exceptions tissue culture systems have relied on the use of primary foreskin fibroblast (HF) cells or primary human embryonic lung cells to study gene expression and virus replication functions. We have observed that primary skin fibroblast (CF) cells derived from the chimpanzee (Pan troglodytes) support the replication of a laboratory strain (Towne) of HCMV. The kinetics of gene expression of the Towne strain grown in CF or HF cells appeared to be equivalent. Titres of progeny virions grown in CF cells appeared to be reduced 10-fold relative to those of virus grown in HF cells. In contrast, replication of the Towne virus was not supported by growth in WES cells (ATCC no. CRL 1609), a chimpanzee skin fibroblast cell line transformed by an adenovirus 12-simian virus 40 hybrid. This study shows that HCMV is less parochial in its host range than previously thought.
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Differential in vitro inhibition of feline enteric coronavirus and feline infectious peritonitis virus by actinomycin D
More LessThe growth of feline enteric coronavirus strain 79-1683 in whole feline embryo cells was inhibited by the presence of 1 µg/ml of actinomycin D in the culture fluid. No virus-specific mRNAs could be detected in such cultures and yields of infectious virus were depressed by >99%. By contrast, the antigenically related feline infectious peritonitis virus strain 79-1146 was unaffected by the presence of actinomycin D, indicating a fundamental difference between the two feline coronavirus strains in their requirements for host-encoded function(s).
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Bovine coronavirus spike glycoprotein: localization of an immunodominant region at the amino-terminal end of S2
More LessWe have identified the binding site of monoclonal antibodies (MAbs) against the S2 subunit of the bovine coronavirus spike (S) glycoprotein. The location of this site was first investigated by using prokaryotic expression of DNA restriction fragments covering the entire S gene. The amino acid sequence containing the antibody binding site was shortened from 70 to 20 amino acids by digestion of plasmid DNA with exonuclease III, followed by sequencing of the smallest digestion product encoding an immunoreactive fusion protein. Finally we synthesized a set of nonapeptides covering the 20 amino acid sequence extending from the N-terminal residue of the S2 subunit (Ala 769 to Tyr 798). MAbs reacted mainly with six consecutive overlapping peptides with the sequence TTGYRFTN-FEPFTV. Polyclonal antibodies from hyperimmunized or convalescent animals reacted only with the recombinant proteins identified by MAbs, and the hyperimmune serum bound to the same set of peptides. This suggests that this highly conserved linear antigenic determinant corresponds to an immunodominant region. This region resembles both in location and immunodominance the linear determinant defined on the infectious bronchitis virus S2 subunit. The presence of similar regions in the N-terminal region of the S2 subunit of other coronaviruses is discussed.
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Sequence analysis of bovine adenovirus type 3 early region 3 and fibre protein genes
More LessThe DNA sequences of the early region 3 (E3) and fibre protein genes of bovine adenovirus type 3 (BAd3) have been determined and the amino acid sequences predicted to be encoded by their open reading frames (ORFs) compared to those of the fibre and E3 proteins from other Ads. One of the BAd3-E3 proteins contains a region homologous to the 14.7K E3 protein of human Ad5 (HAd5). The putative BAd3 fibre protein contains a number of regions homologous to the HAd2 fibre protein sequence, but is predicted to be 244 amino acids longer owing to an increase in the number of repeating structural motifs of hydrophobic amino acid residues in the shaft region. Sequences to the left of the BAd3-E3 gene region contained the 3′ end of another ORF with extensive identity with the hexon-associated protein precursor (pVIII) of HAd2. Like mouse Ad1 and canine Ad1, the BAd3 E3 gene is approximately 1.5 kb, about half the size of the E3 region of HAd2 and HAd5.
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Molecular evidence for the origin of the widespread Venezuelan equine encephalitis epizootic of 1969 to 1972
More LessVenezuelan equine encephalitis (VEE) virus is a mosquito-borne pathogen that has caused encephalitis in equine species and humans during sporadic outbreaks in the western hemisphere. The last, and most widespread, VEE outbreak occurred in South America, Central America, Mexico and the U.S.A. (Texas) during 1969 to 1972. We have cloned and sequenced the genome of a virulent VEE subtype I-AB virus, strain 71-180, isolated in Texas in 1971. Thirty-four nucleotide differences were detected between the genome of 71-180 virus and that of the subtype I-AB Trinidad donkey (TRD) virus isolated during the 1943 VEE epizootic in Trinidad. Fifteen nucleotide changes occurred in the non-structural genes, 16 in the structural genes and three in the 3′ non-coding region. Only six of the nucleotide differences resulted in amino acid substitutions: one change in each of non-structural proteins nsP1 and nsP3, two in the E2 envelope glycoprotein, one in the 6K polypeptide and one in the E1 envelope glycoprotein. The close genetic relationship between 71-180 virus and TRD virus, commonly used for production of formalin-inactivated VEE vaccines, suggests that incompletely inactivated virulent vaccine virus may have been the source of this and other VEE outbreaks. Use of formalized virulent virus was discontinued during the 1969 to 1972 panzootic. No VEE epizootics have been reported since the introduction of the live attenuated TC-83 vaccine virus.
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Extensive antigenic diversification of foot-and-mouth disease virus by amino acid substitutions outside the major antigenic site
More LessThe antigenic sites A and C (the G-H loop and the C terminus, respectively) in VP1 of foot-and-mouth disease virus (FMDV) have been considered the immunodominant regions of the virus involved in the induction of protection. Other antigenic sites have been described but their involvement in protection has not been established. Here we report that two closely related but serologically different FMDVs (the field isolate C3 Argentina/84 and the vaccine strain C3 Resende Br/55) have identical A and C sites but differ at other antigenic sites. Such differences have been documented by reactivity with a panel of 28 monoclonal antibodies (MAbs). The two viruses reacted to the same extent with each of 13 MAbs which recognized epitopes within sites A or C, but reacted differently with six out of 15 MAbs that recognized other sites. Accordingly, sequencing of the entire region coding for the capsid proteins, for both viruses, revealed four amino acid substitutions at three antigenic sites other than A and C. The results suggest that identity of sites A and C may not be sufficient to induce cross-protection, and provide the first evidence of significant antigenic diversification of FMDV in the field mediated by amino acid substitutions outside sites A or C.
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In vitro culture for the detection of infectious human parvovirus B19 and B19-specific antibodies using foetal haematopoietic precursor cells
More LessThe inability to culture human parvovirus B19 in standard cell lines has rendered investigation of clinical samples for the presence of infectious virus problematic. Using haematopoietic precursors derived from first trimester foetal liver as targets for infection, and non-isotopic in situ hybridization to detect intracellular virual DNA, we have assessed infectivity in stored serum samples taken from nine volunteers at different stages following intranasal inoculation with parvovirus B19. Infectious virus was detected as early as 3 days after inoculation, the cessation of infectivity correlating with the rise in specific IgM. In all but two samples, infectivity correlated with the detection of B19 DNA by dot-blot hybridization, although in vitro culture was 10-fold more sensitive than dot-blot hybridization. B19 DNA was detected by the polymerase chain reaction in serum from one volunteer up to 36 days after inoculation, although samples containing specific antibody were non-infectious. Infection of erythroid precursors was completely inhibited by preincubation of virus with serum containing high titre B19-specific IgM and IgG. Unexpectedly, this was associated with a strong B19 DNA hybridization signal within the cytoplasm of phagocytic macrophages. This culture and detection system is a rapid and sensitive means of detecting infectious virus in serum samples, and of assessing the neutralizing ability of B19-specific antibodies.
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Detection of proteinase K-resistant prion protein and infectivity in mouse spleen by 2 weeks after scrapie agent inoculation
More LessThe sequential accumulation of the protease-resistant form of the endogenous prion protein (PrP-res) was compared to levels of scrapie infectivity in the spleen and brain of scrapie-infected mice at various times after inoculation. In mouse spleen PrP-res was detected 1 week after inoculation, and increased 65-fold between 1 and 3 weeks post-inoculation and an additional 15-fold during the next 17 weeks. Infectivity in spleen reached a maximum plateau level by 3 weeks. In contrast, in mouse brain PrP-res was not detected until 8 weeks after inoculation and then increased 200-fold during the next 12 weeks. During this same time, infectivity increased approximately 10000-fold. Therefore, in both spleen and brain of scrapie-infected mice accumulation of PrP-res and infectivity appear to be associated. However, it was not possible to show quantitative correlations between PrP-res detection and infectivity, perhaps owing to the inaccuracy of the infectivity assay.
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Binding of influenza A virus NS1 protein to dsRNA in vitro
More LessThe non-structural protein NS1 of influenza A virus exhibits two modes of RNA-binding activity. One is sequence-specific binding to minus-sense virus RNA with either a 5′- or 3′-terminal common sequence as reported previously. The other was identified as binding to dsRNA and this activity did not show sequence specificity. The affinity of binding to dsRNA was much higher than that to ssRNA. A short miniature virion RNA forming a panhandle structure by pairing between the 5′- and 3′-terminal common sequences bound NS1 with higher affinity and stability than did a dsRNA of similar sequence and length.
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The nucleotide sequence of parsnip yellow fleck virus: a plant picorna-like virus
More LessThe complete sequence of 9871 nucleotides (nts) of parsnip yellow fleck virus (PYFV; isolate P-121) was determined from cDNA clones and by direct sequencing of viral RNA. The RNA contains a large open reading frame between nts 279 and 9362 which encodes a polyprotein of 3027 amino acids with a calculated M r of 336212 (336K). A PYFV polyclonal antiserum reacted with the proteins expressed from phage carrying cDNA clones from the 5′ half of the PYFV genome. Comparison of the polyprotein sequence of PYFV with other viral polyprotein sequences reveals similarities to the putative NTP-binding and RNA polymerase domains of cowpea mosaic comovirus, tomato black ring nepovirus and several animal picornaviruses. The 3′ untranslated region of PYFV RNA is 509 nts long and does not have a poly(A) tail. The 3′-terminal 121 nts may form a stem-loop structure which resembles that formed in the genomic RNA of mosquito-borne flaviviruses.
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The nucleotide sequence of RNA-1 of raspberry bushy dwarf virus
More LessRaspberry bushy dwarf virus (RBDV) has isometric, 33 nm diameter particles and a bipartite RNA genome. Sequencing of the larger component (RNA-1) showed that it consists of 5449 nucleotides and contains one large open reading frame encoding a putative translation product with a calculated M r of 190000. Comparisons of this polypeptide with non-structural proteins of other plant viruses revealed significant homologies with those of alfalfa mosaic virus (AlMV), brome mosaic virus (BMV), cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV). Thus RBDV belongs to the supergroup of ‘Sindbis-like’ plant viruses. The translation product of RBDV RNA-1 contains motifs characteristic of proteins with polymerase, methyltransferase and helicase activities, suggesting that this protein is involved in the replication of the viral RNA. Thus in RBDV, as in TMV, all three functional domains are combined in the single protein, whereas in AlMV, BMV and CMV these domains are distributed over the proteins encoded by RNA-1 and RNA-2. These findings support the idea that RBDV should be placed in a distinct virus genus for which the name idaeovirus has been proposed.
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Resistance to phloem transport of potato leafroll virus in potato plants
More LessA ‘double-graft sandwich’ technique in which sections of potato stem from different potato cultivars were grafted between a susceptible healthy stock plant and a potato leafroll virus (PLRV)-infected scion was used to study the rate of phloem transport of PLRV in cultivars differing in resistance to PLRV infection (IR) and accumulation (AR). Resistance to phloem transport (i.e. delayed PLRV systemic movement) was found in Bismark cultivar (IR AS). This was independent of IR and AR as the rate of movement in Bismark cultivar was markedly slower than that in Omega and Spunta (IR AR), Delaware (IS AR), and Desiree and Renova (IS AS) cultivars. It operated in Bismark cultivar stems of two different ages, but did not operate against potato virus X (PVX) and was not influenced by previous infection with this virus. Aphid vector (Myzus persicae) feeding preferences and colonization rates differed between cultivars, but the cultivar characteristics responsible were unrelated to IR, AR or resistance to phloem transport. Delayed systemic movement of PLRV out of leaves inoculated with viruliferous aphids was independent of AR and resistance to phloem transport, and remained unaffected by previous infection with PVX. It was also independent of cultivar factors causing different aphid feeding preferences and colonization rates, but may be linked to IR.
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The nucleotide sequence of tomato mottle virus, a new geminivirus isolated from tomatoes in Florida
More LessA new geminivirus, tomato mottle virus (TMoV), affecting tomato production in Florida has been cloned and sequenced. Sequence analysis of the cloned replicative forms of TMoV revealed four potential coding regions for the A component [2601 nucleotides (nt)] and two for the B component (2541 nt). Comparisons of the nucleotide sequence of the TMoV genome with those of other whitefly-transmitted geminiviruses indicate that TMoV is a typical bipartite geminivirus of the New World and is closely related to but distinct from abutilon mosaic virus.
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